St. Paul's Cathedral in London had had a boys choir since the 12th century; it was only in the 16th century that the boys of the choir began to act in dramatic performances. Sebastian Westcott was Master of the Children of Paul's in the years 1557–82; in his era, the boys performed 27 times at Court, more than any other troupe, adult or child. Under Master Thomas Giles (1584–99?), the Children of Paul's became closely identified with the plays of John Lyly; they performed at Court nine times in the years 1587–90. The boys acted Lyly's Gallathea at Court, probably on 1 January 1588; they acted his Endymion at Court a month and a day later, on 2 February; and his Midas on 6 January 1590.[1] Other of Lyly's plays, Mother Bombie and Love's Metamorphosis, were also presented at Court in these years.

Also in the 1580s, the Children of Paul's joined the Children of the Chapel in public performances at the first Blackfriars Theatre (1583–4), a foretaste of the period of public performance that was to follow for both companies at the start of the 17th century.

In 1590, however, the Children of Paul's were banned from dramatic performance; they had become involved in the Marprelate controversy through Lyly's actions. For the next ten years the boy companies were out of fashion on the stage.

However, unlike the Children of the Chapel, who worked in the second Blackfriars Theatre, the Children of Paul's had no dedicated theatrical space of their own; when they weren't playing at Court, they acted in the church where they trained as choristers—St. Gregory's Church, just to the southwest of St. Paul's Cathedral. This tended to limit their drama; sometimes plays had to be cut short to accommodate the schedules of the religious institutions in the middle of which the boy players operated.

The Children of Paul's ceased playing around 1606, for unclear reasons. Some scholars have believed that the King's Revels Children, another company that formed c. 1606, might have been, to some significant degree, the Children of Paul's under another name; but this is uncertain. (The King's Revels Children never gelled as an enterprise; they collapsed in litigation among their backers in 1609.)